Immunological memory is a defining feature of the adaptive immune system. Ag-experienced cells respond differently from nave cells. Understanding how such responses differ is of fundamental importance. Yet, there is relatively little knowledge about intrinsic differences between nave and memory cells. Our goal has been to elucidate properties that distinguish memory B cells from their nave precursors. We are approaching this task using a combination of gene expression analysis, functional and biochemical characterization of memory cells and genetic strategies to test the roles of memory-specific molecules in vivo. In the previous period, we overcame a major hurdle to the study of memory B cells by developing a system that creates large numbers of such cells. In this system, the definition of a memory cell is one that had seen and divided in response to Ag and remained as part of an expanded population in a resting state. We first characterized memory cells in this system with phenotypic and functional analysis, then moved to gene expression profiling, identifying multiple genes and proteins that were differentially expressed. These genes highlighted several novel themes with respect to memory B cell function. In addition, we found that memory B cells were comprised of clear phenotypic subsets. We propose first to investigate the ontogeny and functions of these memory B cell subsets. In Aim 1 we will test several hypotheses about the origins of different subtypes of memory B cell. In Aim 2 we will better characterize certain memory B cell subsets and test hypotheses about how they function differently from each other in vitro and in vivo. One theme we discovered in the last period is that B7-family members with inhibitory function are overexpressed on some or all memory B cells. In Aim 3 we use genetic approaches to determine the functions of these molecules on memory B cells, again using both in vitro and in vivo methods. It is quite intriguing that two of the most overexpressed genes we found in memory cells are receptors that are intimately associated with stem cell homeostasis. Though increased in expression by 81- and 15-fold by RT-qPCR, the functions of these genes in the immune system in general and memory B cells in particular are virtually unknown. We hypothesize that these genes play roles in regulating memory cell homeostasis and differentiation, much as they would in other long-lived stem cell populations. In Aim 4 we will used floxed alleles of these genes, along with CD19-Cre mice, and novel mice we recently produced that allow inducible Cre activity specifically in B cells, to determine the functions of these genes in B cell development, responses, and memory formation, as well as in memory cells that were established normally and then induced to delete these genes. Together, these experiments should provide significant advances in an important but relatively understudied area: the origins and function of murine memory B cells.